The broad long-term objective of this research project is to elucidate the structure-function relationships governing the high-affinity streptavidin- biotin interaction. The exceptional affinity of this protein-ligand pair ranks among the highest ever described, and is thus an excellent model system for deriving fundamental insight into the molecular recognition principles underlying high-affinity protein-small molecule interactions. These principles are important for deriving the rules necessary to guide biomedical drug design. The X-ray crystal structure of streptavidin bound to biotin implicates three molecular recognition motifs that are generally important in a number of important protein/enzyme systems. These three classes of interactions have been targeted for the first concerted site- directed mutagenesis, biophysical, and X-ray crystallographic study of streptavidin-biotin interactions. The first class consists of four tryptophan side-chains that mediate aromatic contacts with biotin. Three of these tryptophans have been mutated to alanine and phenylalanine and preliminary X-ray crystallography analysis and characterization of the effects on the binding affinities for biotin and iminobiotin are reported here. Further studies will utilize two additional aliphatic mutants, leucine and methionine, in order to separate the role of the aromatic side-chain from a nonpolar hydrophobic side-chain. Biophysical characterization will include mapping of binding affinity contributions with biotin and iminobiotin, mapping of contributions to the large binding enthalpy using titrating microcalorimetry techniques, and mapping of contributions to the slow dissociation rate that will include measurement of the activation energy for each mutant. The second class consists of an extensive hydrogen-bonding network, and site-directed mutagenesis will again be used to remove the hydrogen-bonding donors and acceptors at the binding site. Additional mutants in this class will target the network at sites removed from the direct donors/acceptors. Similar biophysical studies will be used to quantitatively map the contributions to binding affinity, binding enthalpy, and the off-rate. The third target is the flexible binding loop that becomes ordered upon biotin association. A deletion mutant that removes the loop, and additional mutants that alter the anchoring of the loop hinges will be studied. The effects of these mutations on the binding affinity, binding enthalpy, and the off-rate should provide molecular insight into the role of this flexible binding loop. A crucial component to all of these studies will be concurrent X-ray crystallographic analysis. These studies will provide the structural component necessary to interpret the functional results. With this combination of approaches, the molecular structure-function relationships controlling the high affinity of streptavidin for biotin will be dissected. Because the molecular recognition motifs used by streptavidin are common, the current project will provide useful insight into the fundamental processes used by proteins to generate high affinity.